Development of novel heart failure therapy by using gene transfer into cardiac myocytes

1)将SERCA2基因导入心肌细胞进行心力衰竭治疗的研究我们利用腺相关病毒和Lenti病毒构建了携带人SERCA2基因的基因转移系统。通过将这些重组体感染心肌细胞，我们观察到SERCA2 mRNA和SERCA2蛋白的表达分别增加3倍和2倍。与未携带SERCA2基因的对照组相比，转基因心肌细胞对钙离子的摄取能力提高了40%。我们目前正在研究基因转移对大鼠体内心功能的影响2)通过RNA干扰方法抑制磷蛋白表达来开发心力衰竭治疗方法磷蛋白是SERCA2蛋白钙摄取功能的关键调节蛋白。磷蛋白抑制可增加钙摄取功能。在本研究项目中，我们建立了针对磷蛋白的RNA干扰方法。将磷蛋白基因编码区特异的双链21核苷酸序列导入新生大鼠心肌细胞。磷蛋白siRNA对靶基因的作用具有高度的基因特异性，其基因表达水平仅为对照组的10%。磷蛋白的数量也显著减少到对照的10%。重要的是，钙吸收动力学发生了改变，使效率提高了38%。在过氧化氢诱导的心脏衰竭模型中，磷蛋白RNAi的这些有益作用也得到了证实。在磷蛋白消融组，降低的钙摄取得到恢复。我们的数据表明，钙转运蛋白的基因调控在心力衰竭的治疗中具有一定的疗效。

1) Development of novel heart failure therapy by introducing SERCA2 gene into cardiac myocytesWe have developed gene transfer system bearing human SERCA2 cDNA using adeno-associated virus and lenti-virus. By infecting these constructs into cardiac myocytes, we observed 3-fold increase of SERCA2 mRNA and 2-fold of SERCA2 protein. In the transfected myocytes, Ca2+ uptake capacity was enhanced by 40% in compared with the group infected with control vector that did not harbor SERCA2 cDNA. We are now under investigation of the effect of the gene transfer on the in vivo cardiac function in the rat.2) Development of heart failure therapy by inhibiting the phospholamban expression using a RNA interference methodPhospholamban is a key regulatory protein for Ca2+ uptake function of SERCA2 protein. Inibition of phospholamban increases the Ca2+ uptake function. In this research project, we have developed phospholamban-specific RNA interference method. Double strand 21 ribonucleotide sequence specific for coding region of phospholamban gene was introduced into rat neonatal cardiac myocytes using HVJ envelope. The effect of phospholamban siRNA was highly gene specific for target mRNA and reduced its mRNA level to 10% of control group. The anount of phospholamban protein was also significantly decresed to 10% of control. Importantly, Ca2+ uptake kinetics was shifted to increase the efficiency by 38%. These beneficial effect of phospholamban RNAi was also demonstrated in the hydrogen peroxide-induced failing heart model. Decreased Ca2+ uptake was restored in the phospholamban ablation group.Our data suggest that genetic modulation of Ca2+ transporting protein has a therapeutic benefit in the treatment of heart failure.